Bottom Line:
Higher spontaneous growth of both burst-forming units-erythroid (BFU-E) and CFU-granulocyte/macrophage (CFU-GM) colonies from RAS-mutated patients were observed as compared with control subjects.We also observed a significantly higher amount of GM-colony-stimulating factor-induced p-ERK in children with RASopathies.Collectively, our data provide a basis for further investigating of only partially characterized hematological alterations present in children suffering from RASopathies, and may provide new markers for progression toward malignant MPD in these patients.

ABSTRACTGermline mutations in genes coding for molecules involved in the RAS/RAF/MEK/ERK pathway are the hallmarks of a newly classified family of autosomal dominant syndromes termed RASopathies. Myeloproliferative disorders (MPDs), in particular, juvenile myelomonocytic leukemia, can lead to potentially severe complications in children with Noonan syndrome (NS). We studied 27 children with NS or other RASopathies and 35 age-matched children as control subjects. Peripheral blood (PB) cells from these patients were studied for in vitro colony-forming units (CFUs) activity, as well as for intracellular phosphosignaling. Higher spontaneous growth of both burst-forming units-erythroid (BFU-E) and CFU-granulocyte/macrophage (CFU-GM) colonies from RAS-mutated patients were observed as compared with control subjects. We also observed a significantly higher amount of GM-colony-stimulating factor-induced p-ERK in children with RASopathies. Our findings demonstrate for the first time that PB cells isolated from children suffering from NS or other RASopathies without MPD display enhanced BFU-E and CFU-GM colony formation in vitro. The biological significance of these findings clearly awaits further studies. Collectively, our data provide a basis for further investigating of only partially characterized hematological alterations present in children suffering from RASopathies, and may provide new markers for progression toward malignant MPD in these patients.

Mentions:
A significantly enhanced amount of GM-CSF-stimulated p-ERK-positive cells was observed in monocytes isolated from RAS-mutated patients (n=16) as compared with control subjects (n=19): 28.84±5.192% vs 12.74±3.52% (mean±s.e.m.) P=0.0128, respectively (Figure 3a). This enhanced p-ERK response was also observed at lower dose of GM-CSF (0.1 ng/ml) (4.13±2.34% vs 0.28±0.15%) or under basal conditions (3.39±0.94% vs 1.42±0.54%) in RAS-mutated patients (n=9) as compared with control subject (n=12), although these differences were not statistically significant. When we tested the GM-CSF-induced p-STAT5 response in monocytic cells, we failed to find significant differences between patients (n=13) and control subjects (n=17): 91.15±2.5% vs 84.39±3.63% (mean±s.e.m.) at 0.1 ng/ml GM-CSF and 96.65±0.62% vs 96.91±0.49% at 10 ng/ml GM-CSF (Figure 3b). Finally, IL-6-stimulated p-STAT3 levels were also measured in monocytes from patients (n=15) and controls (n=20). Both subgroups responded to such stimuli in a similar fashion (mean±s.e.m. at 50 ng/ml IL-6: 50.49±7.38% vs 50.30±6.55%, P=NS; Figure 3c).

Bottom Line:
Higher spontaneous growth of both burst-forming units-erythroid (BFU-E) and CFU-granulocyte/macrophage (CFU-GM) colonies from RAS-mutated patients were observed as compared with control subjects.We also observed a significantly higher amount of GM-colony-stimulating factor-induced p-ERK in children with RASopathies.Collectively, our data provide a basis for further investigating of only partially characterized hematological alterations present in children suffering from RASopathies, and may provide new markers for progression toward malignant MPD in these patients.

ABSTRACTGermline mutations in genes coding for molecules involved in the RAS/RAF/MEK/ERK pathway are the hallmarks of a newly classified family of autosomal dominant syndromes termed RASopathies. Myeloproliferative disorders (MPDs), in particular, juvenile myelomonocytic leukemia, can lead to potentially severe complications in children with Noonan syndrome (NS). We studied 27 children with NS or other RASopathies and 35 age-matched children as control subjects. Peripheral blood (PB) cells from these patients were studied for in vitro colony-forming units (CFUs) activity, as well as for intracellular phosphosignaling. Higher spontaneous growth of both burst-forming units-erythroid (BFU-E) and CFU-granulocyte/macrophage (CFU-GM) colonies from RAS-mutated patients were observed as compared with control subjects. We also observed a significantly higher amount of GM-colony-stimulating factor-induced p-ERK in children with RASopathies. Our findings demonstrate for the first time that PB cells isolated from children suffering from NS or other RASopathies without MPD display enhanced BFU-E and CFU-GM colony formation in vitro. The biological significance of these findings clearly awaits further studies. Collectively, our data provide a basis for further investigating of only partially characterized hematological alterations present in children suffering from RASopathies, and may provide new markers for progression toward malignant MPD in these patients.